The invention relates to methods for preparative chiral separation. More particularly, the present invention relates to continuous free-flow electrophoresis preparative chiral separation by addition of chiral auxiliary agent(s).
Separation of enantiomers is an important topic to the pharmaceutical industry. Many of the drugs marketed in the U.S. have at least one chiral center (e.g., ibuprofen and propranolol). Of the 528 synthetically derived chiral drugs, 88% are sold as the racemic mixture. The biological activities and bioavailabilities of the enantiomers sometimes differ. Often, one enantiomer has the desired therapeutic activity whereas the other enantiomer causes undesirable side-effects and may limit drug efficacy or dosage. It is also possible for both enantiomers to have therapeutic value . . . just not for the same disease state. For example, (S)-verapamil is effective as a calcium channel blocker while (R)-verapamil produces cardiac side effects but has potential in reversing multiple drug resistance in cancer therapy. Thus, the development of economical methods for preparative and semipreparative scale chiral separations is highly desirable.
Chiral separations are commonly performed using chiral stationary phases by liquid chromatography (HPLC), first reported in the early 1980""s. The last decade has seen the commercialization of many different types of chiral stationary phases for HPLC. Each of these chiral stationary phases is very successful at separating large numbers of enantiomers which, in many cases, are unresolvable using any other chiral stationary phase. However, a large number of chiral compounds are unresolvable using any of the existing chiral stationary phases. Chiral HPLC columns are more expensive and require more careful handling than conventional columns. Column deterioration often results from a loss of the bonded phase rather than decomposition or racemization of the chiral ligand. Once the chiral column has begun to deteriorate, it generally cannot be returned to its original performance levels. Lot-to-lot variability further hampers method development and large-scale chiral separations remains largely unexplored.
Incomplete understanding of the chiral recognition mechanisms for many of these chiral stationary phases hinders rapid method development. Mobile phase optimization for many chiral HPLC stationary phases is usually not a trivial problem. In conventional HPLC, the relationship between retention and mobile phase composition or column temperature is well-behaved. However, for many chiral stationary phases, normal phase type-behavior may result under very nonpolar mobile phase conditions and reversed-phase behavior under very polar conditions. Often, there is a narrow xe2x80x9cwindowxe2x80x9d of mobile phase conditions under which enantioselectivity is observed and these conditions are usually solute-specific. In contrast to achiral chromatography, there are no chiral TLC plates (except ligand exchange chiral plates) commercially available for scouting mobile phase conditions or likelihood of chiral separation. Thus, column and conditions selection is often reduced to identifying structurally similar analytes for which chiral chromatographic methods have been reported in the scientific literature or chromatographic supply catalogues, and adapting the method for the chiral pair to be resolved.
Preparative chromatography (prep LC), often the method of choice for preparative chiral separations, requires the availability of a suitable chiral stationary phase (CSP). Prep LC columns are also costly and are usually only commercially available on a xe2x80x9cspecial orderxe2x80x9d basis. Lot-to-lot variability in the packing sorbent as well as non-linear adsorption isotherms (e.g., in situ generation of secondary xe2x80x9cchiral phasexe2x80x9d from sorbed enantiomer) arising from mixed mode adsorption complicate scale-up. In addition, the mode of chromatography (e.g., batch vs displacement vs recycling vs simulated moving bed) must be decided.
Bioavailability of drug substances dictates that the compounds be water soluble and many are ionized at physiological pH. The pKa""s of many drugs are well outside the safe operating range for silica-based media (Table 1) and almost all chiral stationary phases currently available are on silica substrates.
Most prep chiral LC employs an organic mobile phase. Many underivatized chiral drugs have only limited solubility in these organic mobile phases. Thus, sample
introduction for many native drugs onto preparative chiral chromatographic columns often requires that packing material be removed from the head of the column, the sample mechanically mixed with this packing material. This mixture is then added to the top of the column, a process which is not easily automated.
Alternative techniques such as counter-current and centrifugal partition chromatographic methods, while allowing chiral selector recovery, require considerable amounts of mobile phase. Stereospecific enzymatic degradation requires identification of a suitable enzyme and often, a complementary enzyme is not available. Enzymatic degradation often preferentially destroys one enantiomer which may have intrinsic value or serve as an internal standard or reference material.
Among the most successful of the liquid chromatographic reversed-phase chiral stationary phases have been the cyclodextrin-based phases. Under predominantly aqueous mobile phase conditions, the mechanism responsible for the chiral selectivity with these phases is thought to rely on inclusion complexation between a hydrophobic moiety of the chiral analyte and the interior of the cyclodextrin cavity. Preferential complexation between one optical isomer and the cyclodextrin results in enantiomeric separation. However, selectivities (xcex1) reported for native cyclodextrin phases in the reversed-phase mode are, in general, less than 2.0 perhaps as a consequence of the low surface concentration of the cyclodextrins (e.g., 0.2-0.3 xcexcmol/m2).
Classically, electrophoresis has been applied to the separation of charged materials such as proteins, nucleic acids, and cells. The separations depend upon differences in charge density and size. Capillary electrophoresis is a well-known technique for the analytical scale separation of chemical components. A sample solution containing molecules to be separated is introduced at one end of a length of capillary tubing containing an electrophoretic medium. Upon application of an electric field across the capillary, different components within the sample migrate at distinct rates towards the oppositely charged end of the capillary dependent upon their relative electrophoretic mobilities in the electrophoretic medium. Due to the varying electromigratory rates, the sample components become increasingly separated into distinct zones or groups as they progress along the capillary. At some position along the capillary, the components of the sample are detected. For example, U.S. Pat. No. 5,061,361 relates to a capillary zone electrophoresis system in which a nanoliter volume of sample is introduced into the capillary tube, and an electric field is imposed on the system to effect separation of the charged components. After migration along the length of the tube, the sample components are detected via ultra-violet absorbance. U.S. Pat. No. 5,084,150 relates to an electrokinetic method of separation in which the surface of moving charged colloidal particles is treated so as to interact selectively with the sample molecules to be separated. The above-described U.S. patents are hereby incorporated by reference.
Recently, capillary electrophoresis (CE) has been shown effective for chiral separations. Chiral separations by CE are usually accomplished using chiral additives in the run buffer. This approach offers several advantages (e.g., additive can be readily changed, a variety of chiral selectors available, rapid screening of chiral selectors, analytes and conditions, small amounts of background electrolyte required, small amounts of chiral additive required, no preequilibration, multiple complexation possible, faster method development than for HPLC). Unfortunately, CE is generally more suited to analytical separations than to preparative scale separations.
Classical gel electrophoresis, a mature method used extensively for protein and nucleic acid purification and characterization has not been routinely used for small molecule separations presumably because small solutes begin to diffuse away from the band center as soon as the applied voltage is removed. Although detection is usually accomplished off-line in electrophoretic and thin layer chromatographic methods, solute affinity for the chromatographic bed and the immediate removal of the mobile phase following the chromatographic run minimizes solute diffusivity in TLC. In contrast to TLC, the gel matrix serves primarily as an anticonvective medium in gel electrophoresis and is designed to minimize interactions with the solute, excluding molecular sieving effects. Hence, there is no mechanism to localize the solute post-run thereby reducing separation efficiency and complicating detection. However, Stalcup et al. demonstrated that analytes complexed with a bulky chiral additive (e.g., sulfated cyclodextrin, MWxcx9c2500), through predominantly electrostatic interactions effectively reduce solute diffusivity to enable enantioseparation using classical gel electrophoresis. Stalcup and co-workers used CE for method development of gel electrophoresis for semi-preparative scale chiral separations.
It should be noted that gel electrophoresis employs less hazardous aqueous solvents than the hydroorganic or organic solvents typically used in most chromatographic-based preparative separations and is less costly in terms of disposal. In addition, costly chiral selectors may be retrieved subsequent to the separation. However, classical gel electrophoresis is a batch process with limited sample throughput.
Preparative continuous free flow electrophoresis translates the tremendous resolving power of electrophoresis into a continuous feed process. Historically, free flow electrophoresis has been used for fractionating charged species such as cells and macromolecules. Free flow electrophoresis is a process in which a sample stream is introduced into a continuous liquid buffer flow at the top of a thin, rectangular electrophoresis chamber while an electric field is imposed perpendicular to the flow within the separation chamber. A fixed or varying electric field is maintained across the separation column perpendicular to the buffer flow. Differential interaction between the various sample components and the electric field produce a lateral displacement of the individual sample components between the two electrodes, dependent upon their charge to weight ratio. Individual sample components can be collected at the opposite end of the chamber using multiple collection ports. Free flow apparatuses are described in U.S. Pat. Nos. 5,562,812, 5,277,774, and 5,082,541, incorporated herein in their entirety.
The angle of the deflection ("THgr") of the solute in the electric field is dependent upon the intrinsic electrophoretic mobility of the solute (xcexci), the linear velocity of the buffer (xcexd) and the current through the chamber (i) and can be described as:                               tan          ⁢                      xe2x80x83                    ⁢          Θ                =                                            μ              i                        ⁢            i                                q            ⁢                          xe2x80x83                        ⁢            κ            ⁢                          xe2x80x83                        ⁢            v                                              (        I        )            
where q is the cross section of the separation chamber and xcexa is the specific conductance of the buffer. The application of (I) to the special case of chiral separations will be discussed in more detail in the Experimental Methods and design.
Despite the use of cooling, microgravitational environments, density and pH gradients, parasitic convection and heat dissipation produced flow stream instability and limited the utility of this approach. However, recent innovations in the design of a continuous free flow electrophoresis apparatus have circumvented the heat dissipation and sample stream distortion inherent in most previous designs. The design exploits the heat exchange capacity of capillary columns by aligning TEFLON capillary tubes close to each other in the electrophoretic chamber. Coolant is pumped through the capillary columns during the electrophoretic run. The system has been used for the separation of biopolymers (e.g., ovalbumin and lysozyme)1 as well as smaller inorganic species (e.g., [CoIII(sepulchrate)]3+ and [CoIII(CN)6]3xe2x88x92). The inclusion of capillary tubes for cooling allows the chamber cross-section to be increased, thereby allowing for fairly high sample throughput. Sample processing rates of 15 mg/hr were reported for a mixture of Amaranth (MW: 804) and Patent Blue VF (MW: 1159).
The magnitude and frequency of the primary electric field, the rate of primary buffer flow, and the frequency of membrane movement are all dependent on the size of the fractionation chamber being used and the electrophoretic mobilities of the species to be separated. Generally, the species being separated are known species so that their mobilities are known. Once a particular size of a fractionation chamber is chosen it is well within the skill of the artisan to optimize the electric field, the rate of primary buffer flow and sample feed to effect the separation of interest.
The present methods disclose a novel approach to preparative chiral separations. The separation of chiral solutes is accomplished according to the present invention by exploiting differences in electrophoretic velocity between chemical species. Complexation between a chiral solute and a chiral additive essentially modifies the intrinsic electrophoretic velocity or mobility of the solute by conferring some of the intrinsic electrophoretic character of the additive on the solute. The extent of electrophoretic mobility modification is dependent upon several factors including the relative sizes and charge densities of the solute and the additive as well as the affinity of the solute for the additive.